The present invention relates to cationic transfection agent(s)/DNA combinations whose particles are stabilized in size with the aid of a nonionic surface-active agent and to their uses in gene therapy.
Numerous genetic diseases are associated with a defect in expression and/or an abnormal, that is to say a deficient or excessive, expression of one or more nucleic acids. The main objective of gene therapy is to correct this type of genetic abnormality through cellular expression in vivo or in vitro of cloned genes.
Nowadays, several methods are proposed for the intracellular delivery of this type of genetic information. One of them, in particular, is based on the use of chemical or biochemical vectors. Synthetic vectors have two main functions: complexing the DNA to be transfected and promoting its cellular attachment as well as its passage across the plasma membrane and, where appropriate, across the two nuclear membranes. Among the synthetic vectors developed, the cationic polymers of the polylysine and DEAE-dextran type or alternatively lipofectants are the most advantageous.
A major advance was accomplished in this mode of transfection with the development of a technology based on the use of cationic transfection agents of the lipofectant type and more precisely of cationic lipids. It has thus been demonstrated that a positively charged cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) interfered, in the form of liposomes or of small vesicles, spontaneously with DNA, which is negatively charged, to form lipid-DNA complexes capable of fusing negatively charged, to form lipid-DNA complexes capable of fusing with the cell membranes, and thus allowed the intracellular delivery of the DNA.
Since DOTMA, other cationic lipids have been developed on this model of structure: lipophilic group combined with an amino group via a “spacer” arm. Among these, there may be mentioned more particularly those comprising, as lipophilic group, two fatty acids or a cholesterol derivative, and comprising, in addition, where appropriate, as amino group, a quaternary ammonium group. DOTAP, DOBT or ChOTB may be mentioned in particular as representatives of this category of cationic lipids. Other compounds, such as DOSC and ChOSC, are characterized by the presence of a choline group in place of the quaternary ammonium group.
Another category of lipofectants, the lipopolyamines, has also been described. In general, it is an amphiphilic molecule comprising at least one hydrophilic polyamine region combined, through a so-called spacer region, with a lipophilic region. The polyamine region of lipopolyamines, which is cationically charged, is capable of reversibly combining with the nucleic acid, which is negatively charged. This interaction greatly compacts the nucleic acid. The lipophilic region makes this ionic interaction insensitive to the external medium by coating the nucleolipid particle formed with a lipid film. In this type of compounds, the cationic group may be represented by the radical L-5-carboxyspermine which contains four ammonium groups, two which are primary and two which are secondary. The compounds DOGS and DPPES belong thereto. These lipopolyamines are most particularly effective for the transfection of primary endocrine cells. As representative of this last family of compounds, there may be mentioned more particularly the lipopolyamines described in particular in Patent Applications WO 96/17823 and WO 97/18185.
However, the efficacy of these synthetic vectors remains to be improved particularly in terms of formation of a complex with nucleic acid and more precisely from the point of view of the stability of the particles of these nucleolipid complexes. Indeed, with conventional nucleic acid/cationic transfection agent formulations, a phenomenon of aggregation of the particles of complexes is frequently and rapidly observed. Such aggregates obviously possess a size which is hardly compatible with a therapeutic transfection. One of the solutions proposed up until now for overcoming this type of precipitation phenomenon consists in introducing into the formulation the cationic transfection agent, such as for example lipofectant, in an excessive quantity, that is to say in a lipofectant/nucleic acid charge ratio of the order of 10 or more. In addition to the fact that such a solution is not always effective, it is not completely satisfactory from the point of view of safety. Cationic transfection agents such as lipofectants and cationic polymers are in themselves compounds which, in large quantities, risk exhibiting a relative toxicity for the cells incorporating them.
It would therefore be particularly advantageous to have from the therapeutic point of view cationic transfection agent/nucleic acid formulations possessing reduced charge ratios and nevertheless stabilized over time in the form of nonaggregated particles. However, as stated above, the nucleic acid/lipofectant concentration zones corresponding to such charge ratios are generally associated with an unstable physical state. A phenomenon of aggregation of the nucleolipid particles is rapidly observed. Furthermore, it is also known that the presence of a salt of the NaCl type, which is conventionally used in cationic transfection agent/nucleic acid formulations, can induce, at certain concentrations, the precipitation of the nucleolipid particles. Thus, the greater the salt concentration, the greater the transfection agent concentration range for which the precipitation is observed.